Serine proteases serve an important role in human physiology by mediating the activation of vital functions. In addition to their normal physiological function, serine proteases have been implicated in a number of pathological conditions in humans. Serine proteases are characterized by a catalytic triad consisting of aspartic acid, histidine and serine (Asp-His-Ser) at the active site.
The naturally occurring serine protease inhibitors are usually, but not always, polypeptides and proteins which have been classified into families primarily on the basis of the disulfide bonding pattern and the sequence homology of the reactive site. Serine protease inhibitors (serpins) have been found in microbes, in the tissues and fluids of plants, animals, insects and other organisms. Protease inhibitor activities were first discovered in human plasma by Fermi and Pernossi in 1894. At least nine separate, well-characterized proteins are now identified, which share the ability to inhibit the activity of various proteases. Several of the inhibitors have been grouped together, namely alpha-1-proteinase inhibitor, antithrombin III, antichymotrypsin, C1-inhibitor, eglin, and alpha-2-antiplasmin, which are directed against various serine proteases, i.e., leukocyte elastase, thrombin, cathepsin G, chymotrypsin, plasminogen activators, and plasmin. These are referred to as the alpha-1-proteinase inhibitor class. The protein alpha-2-macroglobulin inhibits members of all four catalytic classes: serine, cysteine, aspartic, and metalloproteases. However, other types of protease inhibitors are class specific. The alpha-1-proteinase inhibitor (also known as α1-antitrypsin or AAT) and inter-alpha-trypsin inhibitor inhibit only serine proteases, alpha-1-cysteine protease inhibitor inhibits cysteine proteases, and alpha-1-anticollagenase inhibits collagenolytic enzymes of the metalloenzyme class.
AAT is a glycoprotein of MW 51,000 with 394 amino acids and 3 oligosaccharide side chains. Human AAT was named anti-trypsin because of its initially discovered ability to inactivate pancreatic trypsin. Human AAT is a single polypeptide chain with no internal disulfide bonds and only a single cysteine residue normally intermolecularly disulfide-linked to either cysteine or glutathione. The reactive site at position 358 of AAT contains a methionine residue, which is labile to oxidation upon exposure to tobacco smoke or other oxidizing pollutants. Such oxidation may reduce the biological activity of AAT; therefore substitution of another amino acid at that position, i.e. alanine, valine, glycine, phenylalanine, arginine or lysine, produces a form of AAT which is more stable. AAT can be represented by the following formula: MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAF SLYRQLASTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLR TLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDY VEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKV PMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLE NEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHK AVLTID EKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO: 19). The amino acid sequence of human alpha-1 antitrypsin can be that of SEQ ID NO:20.
(Details of the sequence can be found for example in U.S. Pat. No. 5,470,970, incorporated herein by reference in its entirety).
The C-termini of human antitrypsin (AAT), is homologous to antithrombin (ATIII), antichymotrypsin (ACT), C1-inhibitor, tPA-inhibitor, mouse AT, mouse contrapsin, barley protein Z, and ovalbumin. Its normal plasma concentration ranges from 1.3 to 3.5 mg/ml although it can behave as an acute phase reactant by increasing 3-4-fold during host response to inflammation and/or tissue injury such as with pregnancy, acute infection, and tumors. Alpha-1-antitrypsin, known to be an acute phase protein in humans, is augmented in autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), mixed connective tissue disease (MCTD), Sjogren syndrome, scleroderma, and other sclerotic diseases. AAT may play an important role as an early marker for the diagnosis of such autoimmune disorders.
AAT easily diffuses into tissue spaces and forms a 1:1 complex with a target protease, principally neutrophil elastase. Human neutrophil elastase (NE) is a proteolytic enzyme secreted by polymorphonuclear leukocytes in response to a variety of inflammatory stimuli. The degradative capacity of NE, under normal circumstances, is modulated by relatively high plasma concentrations of α1-antitrypsin (AAT). However, stimulated neutrophils produce a burst of active oxygen metabolites, some of which (hypochlorous acid for example) are capable of oxidizing a critical methionine residue in AAT. Oxidized AAT has been shown to have a limited potency as a NE inhibitor and it has been proposed that alteration of this protease/antiprotease balance permit NE to perform its degradative functions in a non-localized and uncontrolled fashion.
Other enzymes such as trypsin, chymotrypsin, cathepsin G, plasmin, thrombin, tissue kallikrein, and factor Xa can also serve as substrates. The enzyme/inhibitor complex is removed from circulation by binding to serpin-enzyme complex (SEC) receptor and catabolized by the liver and spleen cells. Humans with circulating levels of AAT less than 15% of normal are susceptible to the development of lung disease, e.g., familial emphysema, at an early age. Therefore, it appears that this inhibitor represents an important part of the defense mechanism against attack by serine proteases.
In some instances the degradative action of serine proteases results in serious pathological conditions or disease states. For example, elastase is a protease which causes degradation and fragmentation of elastic fibers as a result of its proteolytic activity on rubber-like elastin. Other connective tissue proteins, such as type I, III, and IV collagens, the protein portion of proteoglycans, and laminin may be also cleaved by elastase. Tissues comprising the lungs, bronchi, ear, and skin contain large amounts of elastin. Excessive degradation of elastin has also been associated with arthritis, atherosclerosis, certain skin diseases, pulmonary emphysema and acute respiratory-distress syndrome. Therefore, by inhibiting the activity of elastase it is possible to treat a wide variety of pathological conditions including pulmonary emphysema, various clotting disorders and inflammatory processes.
One illustration of the importance of the catalytic activity of serine proteases is provided by the role of human neutrophil elastase and one of its natural inhibitors, AAT, in the pathogenesis of emphysema or cystic fibrosis. In the lungs of healthy individuals there is a balance between the levels of elastase and its inhibitors. The elastase serves in the repair and turnover of connective tissues (elastin) and the AAT is involved in the regulation and clearance of elastase. Disruption of the elastase/AAT balance leads to increased elastin degradation and, hence, to elastic tissue destruction. A prolonged imbalance leads to an irreversible dilation of pulmonary airways and damage to the respiratory tissues of the lung, a condition known as pulmonary emphysema. As another example, oxidants from the condensate of cigarette smoke have been shown to drastically reduce the elastase binding affinity of AAT by oxidizing a methionine residue within the reactive site. A final example involves both elevated levels of elastase and simultaneously lower levels of functional AAT inhibitor. The inflammatory response to foreign particulate matter or cigarette smoke leads to elevated levels of polymorphonuclear leukocytes in the lungs. These cells disrupt the protease/protease inhibitor balance by secretion of proteolytic enzymes, e.g., elastase. They also secrete oxidants including myeloperoxidase which appear to oxidatively inactive AAT.
So far, AAT is one of few naturally-occurring mammalian serine protease inhibitors clinically approved for the therapy of protease imbalance. Therapeutic AAT became commercially available in the mid 80's and is prepared by various purification methods (see for example Bollen et al., U.S. Pat. No. 4,629,567; Thompson et al., U.S. Pat. No. 4,760,130; U.S. Pat. No. 5,616,693; WO 98/56821). PROLASTIN® is a trademark for a purified variant of AAT, is currently sold by Bayer Company (U.S. Pat. No. 5,610,285 Lebing et al., Mar. 11, 1997). Recombinant unmodified and mutant variants of AAT produced by genetic engineering methods from transformed cells are also known (U.S. Pat. No. 4,711,848); methods of delivery are also known, e.g., AAT gene therapy/delivery (U.S. Pat. No. 5,399,346 to French Anderson et al.).
Human Immunodeficiency Virus (HIV)
The replication of HIV requires protease activity required for the cleavage of gag-pol precursor proteins. This enzymatic activity is similar to activity of renin-aspartyl protease produced by the kidney. The close relationship between renin and HIV encoded protease led to an accelerated generation of specific HIV-1 protease inhibitors as effective agents in treatment of AIDS (Scharpe, et al., “Proteases and their inhibitors: today and tomorrow”, Biochimie, 73(1):121-6 (1991). Many therapeutic agents directed against HIV protease have been developed as a consequence and used successfully in AIDS patients. For example, indinavir and crixivan are aspartyl protease inhibitors, which inhibit cleavage of pre-protein of HIV by viral own protease and thereby suppress viral proliferation. Lezdey et al., (U.S. Pat. No. 5,532,215) disclose the method of using AAT, Secretory Leukocyte Protease Inhibitor (SLPI), and alpha antichymotrypsin (AAC) for inhibition of proliferation of a variety of viruses that require gag-pol cleavage. They claim that AAT, SLPI, and AAC, generally known as serine protease inhibitors, inhibit such viruses by binding to viral or cellular aspartic protease. While it is unknown whether this mechanism may take place in such circumstances, several lines of evidence exist, which indicate that serine protease inhibitors may interfere with viral replication through inhibition of host's serine proteases but not HIV encoded aspartyl protease.
Several serine proteases of the human host have been identified in the past as being involved in HIV infection. Investigators argued that the endoproteolytic cleavage of the envelope glycoprotein precursor (gp160) of the HIV by a cellular protease is required for full activation of the virus. The first one, so-called Kunitz-type basic proteinase or tryptase TL2, was proposed by Kido et al., “A novel membrane-bound serine esterase in human T4+ lymphocytes immunologically reactive with antibody inhibiting syncytia induced by HIV-1. Purification and characterization”, J Biol Chem., 15; 265(35):21979-85 (1990); and Brinkmann et al., “Inhibition of tryptase TL2 from human T4+ lymphocytes and inhibition of HIV-1 replication in H9 cells by recombinant aprotinin and bikunin homologues”, J Protein Chem, 16(6):651-60), (1997). Accordingly, the recombinant (K15R M52E) aprotinin—a Kunitz-type inhibitor—reduced HIV-1 replication in H9 cells at a concentration of 50 microM. (Auerswald et al., “K15R M52E) aprotinin is a weak Kunitz-type inhibitor of HIV-1 replication in H9 cells” Biomed Biochim Acta, 50(4-6):697-700 (1991)).
A calcium-independent processing protease, viral envelope glycoprotein maturase (VEM), converted HIV envelope glycoprotein precursor gp160 to gp120 and gp41 and was identified by Kamoshita et al., (Kamoshita et al., “Calcium requirement and inhibitor spectrum for intracellular HIV type 1 gp160 processing in cultured HeLa cells and CD4+ lymphocytes: similarity to those of viral envelope glycoprotein maturase”, J Biochem, June; 117(6): 1244-53) (Tokyo 1995).
A neutralizing epitope of HIV on external envelope glycoprotein (gp120) was found to have homologous sequences to inter-alpha-trypsin inhibitor (ITI). Human urinary trypsin inhibitor (UTI, a protein indistinguishable from ITI, as well as synthetic peptides including epitope beta inhibited syncytium formation caused by the HIV-infected CCRF-CEM and uninfected Molt-4 cells in a dose-dependent manner (0.1-1 mM). These findings suggested that epitope on gp120 could be a substrate for trypsin-like protease upon HIV-1 infection (Koito et al., “A neutralizing epitope of human immunodeficiency virus type 1 has homologous amino acid sequences with the active site of inter-alpha-trypsin inhibitor”, Int Immunol, 1(6):613-8) (1989).
A naturally occurring serine protease inhibitor or serpin, secretory leukocyte protease inhibitor (SLPI) was shown to inhibit HIV in monocytic cells. SLPI did not appear to act on the virus directly, but rather through interaction with the host cell (McNeely et al., “Secretory leukocyte protease inhibitor: a human saliva protein exhibiting anti-human immunodeficiency virus 1 activity in vitro”, J Clin Invest, 96(1):456-64) (1995).
Hallenberger et al., identified the serine protease furin, which recognizes the amino-acid sequence Arg-X-Lys/Arg-Arg as a cleavage site, as involved in HIV infection (Hallenberger et al., “Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160”, Nature, 26; 360(6402):358-61) (1992). In addition to furin, other subtilisin/kexin-like convertases including PACE4, PC5/6-B and PC1 were also proposed as candidate enzymes and the co-expression of the [Arg355, Arg358]-alpha-1-antitrypsin—furin-directed Portland variant—was shown to potently inhibit the processing of both gp160 and gp120 by these convertases (Vollenweider, et al., “Comparative cellular processing of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp160 by the mammalian subtilisin/kexin-like convertases”, Biochem, 1; 314 (Pt 2):521-32) (1996). Another mutant variant of AAT, directed against furin, was recently proposed as a specific HIV inhibitor (Anderson et al., “Inhibition of HIV-1 gp160-dependent membrane fusion by a furin-directed alpha 1-antitrypsin variant”, J Biol Chem, 268(33):24887-91(1993); and also U.S. Pat. No. 5,604,201, incorporated herein by reference in its entirety).
Meanwhile, Decroly et al., believe that kexin/subtilisin-related endoproteases including furin, PC5/6, and the newly cloned PC7 (LPC/PC7) are main convertase enzyme candidates responsible for the cleavage of the HIV envelope glycoprotein (Decroly, et al., “Identification of the paired basic convertases implicated in HIV gp160 processing based on in vitro assays and expression in CD4(+) cell lines”, J Biol Chem, 271(48):30442-50) (1996).
A human analogue of endoprotease Kex2p, from the yeast Saccharomyces cerevisiae, was proposed as a cellular enzyme processing HIV envelope glycoprotein precursor (Moulard, et al., “Kex2p: a model for cellular endoprotease processing human immunodeficiency virus type 1 envelope glycoprotein precursor”, Eur J Biochem, 225(2):565-72 (1994); Franzusoff, et al., “Biochemical and genetic definition of the cellular protease required for HIV-1 gp160 processing”, J Biol Chem, 270(7):3154-9) (1994). These serine proteases when expressed within the host cell were postulated to operate not only on the cell surface but also intracellularly.
A cathepsin G-like proteinase at the surface of U-937 cells reacting with the V3 loop of HIV-1 gp120 was reported by Avril et al., (Avril, et al., “Identification of the U-937 membrane-associated proteinase interacting with the V3 loop of HIV-1 gp120 as cathepsin G”, FEBS Lett, 345(1):81-6) (1994).
At least five separate T lymphocyte-derived enzymes, mostly zinc-dependent metalloproteinases with affinity to HIV envelope, were identified by Harvima et al., (Harvima et al., “Separation and partial characterization of proteinases with substrate specificity for basic amino acids from human MOLT-4 T lymphocytes: identification of those inhibited by variable-loop-V3 peptides of HIV-1 (human immunodeficiency virus-1) envelope glycoprotein”, Biochem J, 292 (Pt 3):711-8) (1993).
Acrosin, a serine protease from semen, was also identified as being involved in HIV infection (Bourinbaiar, et al., “Acrosin inhibitor, 4′-acetamidophenyl 4-guanidinobenzoate, an experimental vaginal contraceptive with anti-HIV activity”, Contraception, 51(5):319-22) (1995).
T lymphocyte associated elastase was reported by Bristow et al., as a protease involved in HIV infection and synthetic elastase inhibitors MAAPVCK but not FLGFL were shown to interfere with HIV infection (Bristow, et al., “Inhibition of HIV-1 by modification of a host membrane protease”, Int Immunol, 7(2):239-49) (1995).
Human proteases PC6A and PC6B isomers were also proposed as gp160 processing enzymes (Miranda et al., “Isolation of the human PC6 gene encoding the putative host protease for HIV-1 gp160 processing in CD4+ T lymphocytes”, Proc Natl Acad Sci USA, 93(15):7695-700) (1996).
A major serine protease found in plasma, adequately called plasmin, was recently found to be involved in gp160 cleavage (Okumura et al., “The extracellular processing of HIV-1 envelope glycoprotein gp160 by human plasmin”, FEBS Lett, 442(1):39-42) (1998).
The V3 loop of gp120 was found to be homologous with trypstatin and peptides mimicking V3 region were found to inhibit HIV infection (Cox et al., “Synergistic combinations and peptides in the inhibition of human immunodeficiency virus”, Adv Enzyme Regul, (31:85-97) (1991).
Several other cellular endoproteases were proposed in the course of the last few years to be involved with HIV but their identity is still unknown (Bukrinskaia et al., “Inhibition of HIV reproduction in cultured cells using proteolysis inhibitors”, Vopr Virusol, 34(1):53-5 (1989); Avril et al., “Interaction between a membrane-associated serine proteinase of U-937 monocytes and peptides from the V3 loop of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein”, FEBS Lett, 317(1-2):167-72 (1993); Bourinbaiar, et al., “Effect of serine protease inhibitor, N-alpha-tosyl-L-lysyl-chloromethyl ketone (TLCK), on cell-mediated and cell-free HIV-1 spread”, Cell Immunol, 155(1):230-6 (1994); Schwartz, et al., “Antiviral activity of the proteasome on incoming human immunodeficiency virus type 1”, J Virol, 72(5):3845-50) (1998).
Thus the list of serine proteases as HIV facilitating enzymes has gradually increased and today in addition to TL2 it includes an assortment of enzymes including furin, kexin, convertase, cathepsin G, subtilisin, subtilisin-like proteases, tryptase M, acrosin, PACE4, PC5/6-B, PC1, VEM, etc.
Although mainstream AIDS research is still concentrated on inhibitors of HIV encoded aspartyl protease, considerable work is being conducted primarily aimed at identifying host cellular endoproteases. While some earlier reports identifying various proteases such as Kunitz type tryptase TL2 seem to have been confirmed, other enzymes as facilitators of HIV infection failed to pass rigorous scientific scrutiny.
For example, furin was found important but not essential for the proteolytic maturation of gp160 of HIV-1 (Ohnishi et al., “A furin-defective cell line is able to process correctly the gp160 of human immunodeficiency virus type 1”, Virol, 68(6):4075-99 (1994); Gu et al., “Furin is important but not essential for the proteolytic maturation of gp160 of HIV-1”, FEBS Lett, 365(1):95-7) (1995); Inocencio et al., “Endoprotease activities other than furin and PACE4 with a role in processing of HIV-1 gp160 glycoproteins in CHO-K1 cells”, J Biol Chem, 272(2): 1344-8) (1997).
Similarly, the inhibition of HIV with saliva-derived SLPI as originally reported by McNeely et al., was not supported by subsequent research in several independent labs (Turpin et al., “Human immunodeficiency virus type-1 (HIV-1) replication is unaffected by human secretory leukocyte protease inhibitor”, Antiviral Res, 29(2-3):269-77 (1996); Kennedy et al., “Submandibular salivary proteases: lack of a role in anti-HIV activity”, J Dent Res, 77(7): 1515-9) (1998).
The anti-HIV effect of AAT as speculated by Lezdey et al., (U.S. Pat. No. 5,532,215, incorporated herein by reference in its entirety) was also not confirmed by actual experimental studies carried out by practitioners in the art. Two separate studies, one conducted by Anderson et al., (J Biol Chem, 268(33):24887-91 (1996); and other by Vollenweider et al., (Biochem J, 314 (Pt 2):521-32) (1996), have convincingly demonstrated that naturally occurring or non-mutated AAT directed against its natural substrate, elastase, has not shown any anti-HIV activity. Similarly Harvima et al., have shown that putative tryptase receptors on T lymphocytes were not reactive with anti human anti-tryptase antibody (Harvima et al., “Separation and partial characterization of proteinases with substrate specificity for basic amino acids from human MOLT-4 T lymphocytes: identification of those inhibited by variable-loop-V3 peptides of HIV-1 (human immunodeficiency virus-1) envelope glycoprotein”, Biochem J, 292 (Pt 3):711-8) (1993). Furthermore, Meylan et al., stated that AAT natural substrates such as trypsin, factor Xa, and mast cell tryptase did not enhance the HIV infectivity (Meylan et al., “HIV infectivity is not augmented by treatment with trypsin, Factor Xa or human mast-cell tryptase”, AIDS, 6(1):128-30) (1992).
As a result of enzyme studies pertaining to HIV replication, numerous serine protease inhibitors were identified. These include transition state analog peptides such as decanoyl-Arg-Lys-Arg-Arg-psi [CH2NH]-Phe-Leu-Gly-Phe-NH2, substrate analogues such as decanoyl-RVKR-chloromethylketone, suicide substrates such as diisopropyl fluorophosphate (DFP), microbial inhibitors like leupeptin and antipain, leech-derived recombinant tryptase inhibitor (Auerswald et al., “Recombinant leech-derived tryptase inhibitor: construction, production, protein chemical characterization and inhibition of HIV-1 replication”, Biol Chem Hoppe Seyler, 375(10):695-703) (1994), eglin, trypsin-type protease inhibitors aprotinin, HI-30, E-64, trypstatin, bikunin, H130, N-alpha-tosyl-L-lysyl-chloromethyl ketone, aryl guanidinobenzoates, MG132, and lactacystin (a complete list of inhibitors can be gleaned from the references identified supra, which are herein incorporated by reference in their respective entireties). Yet, despite all these efforts not a single compound has been considered as clinically acceptable. This is mainly due to the fact that serine protease inhibitors in general have a broad inhibitory range not only toward HIV facilitating enzymes but also against vital proteolytic enzymes that are necessary for a normal function of a host.
In the course of the AIDS progression, many measurable clinical parameters including AAT progressively increase (Cordiali Fei et al., “Behavior of several ‘progression markers’ during the HIV-Ab seroconversion period. Comparison with later stages”, J Biol Regul Homeost Agents, 6(2):57-64) (1992). AAT was positive in 40% of HIV-positive patients with cryptosporidial infections and none of 12 HIV-positive patients with non-cryptosporidial diarrhea (Lima et al., “Mucosal injury and disruption of intestinal barrier function in HIV-infected individuals with and without diarrhea and cryptosporidiosis in northeast Brazil”, Am J Gastroenterol, 92(10): 1861-6) (1997). Serum concentrations of a tumor-associated trypsin inhibitor (TATI) were very high in some HIV positive subjects and especially in AIDS patients (Banfi et al., “Tumor-associated trypsin inhibitor in induced and acquired immunodeficiency. Studies on transplanted and HIV-infected patients”, Scand J Clin Lab Invest Suppl, 207:55-8 (1991)). The incidence of abnormal AAT phenotypes was 16.3% in the homosexual group which was significantly different (p less than 0.03) than the 8.7% in the heterosexual group. There was no difference in the phenotype distribution between homosexuals who were anti-HIV antibody reactive and those who were non-reactive (Deam et al., “Alpha 1-antitrypsin phenotypes in homosexual men”, Pathology, 21(2):91-2) (1989). Faecal alpha1 antitrypsin concentration were reflective of abnormal pancreatic function of paediatric HIV infection (Carroccio et al., “Pancreatic dysfunction and its association with fat malabsorption in HIV infected children”, Gut, 43(4):558-63) (1998). Patients with HIV-1 infection are known to acquire an obstructive pulmonary disease with clinical similarity to emphysema. AAT levels measured in these patients were in the lower normal range. Despite observing these clinical findings in 42% of consecutive HIV-1-infected patients in the clinic, no evidence of current of previous opportunistic infection was detected. Bronchoalveolar lavage fluid obtained in a subset of these patients contained TNF and free radicals, indicating inflammation. It is possible that HIV-1 associated free radical production inactivated pulmonary AAT and facilitated the development of the cryptogenic emphysema-like condition.
Also, high levels of serum trypsin and elastase are present in an elevated percentage of patients with AIDS, suggesting that the pancreas is frequently damaged in this disease. A significant inverse relationship was found between serum enzyme concentrations and the number of CD4+ lymphocytes (Pezzilli et al., “Serum pancreatic enzymes in HIV-seropositive patients”, Dig Dis Sci, 37(2):286-8) (1992).
In vitro studies have shown that HIV-1 was found sensitive to inactivation by low concentrations of trypsin (Tang et al., “Inactivation of HIV-1 by trypsin and its use in demonstrating specific virus infection of cells”, J Virol Methods 33(1-2):39-46) (1991). This led to the belief that trypsin therapy might be useful to treat HIV. Chymotrypsin and trypsin manufactured in the former USSR in doses of 10 mg each administered intramuscularly appeared to normalize the abnormal, reduced ratio of CD4/CD8 cells—a condition observed in persons infected with the HIV (Glozman, “Immunologic foundation of enzyme therapy of patients with orchiepididymitis”, Antibiot Khimioter, 35(7):50-2) (1990).
Prior to the present invention it was generally believed that the naturally occurring serine protease inhibitor AAT was ineffective against HIV infection. Alternatively, prior to the present invention it was speculated that AAT might be useful for inhibition of HIV proliferation by blocking HIV encoded aspartyl protease or a similar cellular protease that mediate gag-pol cleavage. The present inventor has discovered that, contrary to these earlier convictions, naturally occurring AAT and derivatives thereof are useful for inhibition of HIV via several unexpected modes of action not recognized in the prior art.
It is therefore the goal of the present invention, in its broadest aspect, to provide methods of treating diseases dependent on the action of protease inhibitors. Accordingly, it should be recognized that this invention is applicable to the control of catalytic activity of serine proteases in any appropriate situation including, but not necessarily limited to, medicine, biology, agriculture, and microbial fermentation. These and other objects and advantages of the present invention will be recognized by those skilled in the art from the following description and representative examples.
It is therefore the overall object of the present invention to provide compounds, which exhibit inhibitory activity toward serine proteases.
It is an object of the present invention to provide clinically acceptable serine protease inhibitors with recognized utility and exhibiting relatively high activity at relatively low concentrations.
It is another object of the present invention to provide serine protease inhibitors exhibiting selectivity for certain key proteases involved in viral activation and infection. It is yet another object of the invention to provide means of regulating virus release by compounds having AAT activity either alone or in combination with other anti-HIV compounds.
These and other objects and advantages of the present invention will be recognized by those skilled in the art from the following description and illustrative examples.
The present invention offers useful insight into therapy and pathogenesis of viral infection. In particular it provides a method of treating viral infection facilitated or mediated by a serine proteolytic (SP) activity comprising administering to a subject suffering or about to suffer from said viral infection a therapeutically effective amount of a compound having a serine protease inhibitory or serpin activity comprising α1-antitrypsin activity (AAT). The viral infection may include retroviral infection such as human immunodeficiency virus (HIV) infection.
A method of preventing or inhibiting delivery of viral nucleic acid into the nucleus of a mammalian host as well as a method of preventing or inhibiting the exit of a virion particle from a mammalian host harboring an agent of a viral infection is provided. Preferably these processes are mediated by endogenous host serine protease (SP) or SP-like activity and will be counteracted by administering a pharmacologically effective amount of a substance exhibiting mammalian α1-antitrypsin (AAT) or AAT-like activity. According to this method the post-exposure prophylaxis is contemplated in order to block establishment of productive infection in a mammal exposed to HIV-contaminated fluids such as blood, saliva, semen, sweat, urine, vaginal secretion, tears, and other body fluids that may contain HIV either in cell-free form or in cell-associated from. It also understood that said method is effective in preventing mother-to-child HIV transmission during pregnancy. According to this method pharmacologically effective amount of a substance exhibiting mammalian α1-antitrypsin (AAT) or AAT-like activity incorporated in topical vaginal or rectal formulations as well as in condoms and intrauterine devices (IUD) is useful for preventing sexual transmission of HIV.
Among preferred compounds to treat such viruses is a substantially purified natural or recombinant AAT. AAT and similarly active compounds may be identified by a series of assays wherein a compound (AAT) will exhibit inhibitory activity versus control in an assay. One of these assays comprises blocking interleukin-18 or IL-18-induced human immunodeficiency virus (HIV) production in U1 monocytic cells. Other assays involve blocking stimulants such as IL-6, NaCl, LPS, TNF, and other HIV stimulants known in the art. Other assays involve MAGI-CCR-5 cell assay and PBMC assay as described in detail in the body of the disclosure.
Also contemplated is a series of peptides comprising carboxyterminal amino acid peptides corresponding to AAT. (The Sequence Listing included herein denotes F=X, see listing) Among this series of peptides, several are equally acceptable including FVFLM (SEQ ID NO: 1), FVFAM (SEQ ID NO: 2), FVALM (SEQ ID NO: 3), FVFLA (SEQ ID NO: 4), FLVFI (SEQ ID NO: 5), FLMII (SEQ ID NO: 6), FLFVL (SEQ ID NO: 7), FLFVV (SEQ ID NO: 8), FLFLI (SEQ ID NO: 9), FLFFI (SEQ ID NO: 10), FLMFI (SEQ ID NO: 11), FMLLI (SEQ ID NO: 12), FIIMU (SEQ ID NO: 13), FLFCI (SEQ ID NO: 14), FLFAV (SEQ ID NO: 15), FVYLI (SEQ ID NO: 16), FAFLM (SEQ ID NO: 17), AVFLM (SEQ ID NO: 18), and combination thereof.
These pentapeptides can be represented by a general formula (1): I-A-B-C-D-E-F-G-H-II, wherein I is Cys or absent; A is Ala, Gly, Val or absent; B is Ala, Gly, Val, Ser or absent; C is Ser, Thr or absent; D is Ser, Thr, Ans, Glu, Arg, Ile, Leu or absent; E is Ser, Thr, Asp or absent; F is Thr, Ser, Asn, Gln, Lys, Trp or absent; G is Tyr or absent; H is Thr, Gly, Met, Met(O), Cys, Thr or Gly; and II is Cys, an amide group, substituted amide group, an ester group or absent, wherein the peptides comprise at least 4 amino acids and physiologically acceptable salts thereof.
The peptides of interest are homologous and analogous peptides. While homologues are natural peptides with sequence homology, analogues will be peptidyl derivatives, e.g., aldehyde or ketone derivatives of such peptides. Typical examples of analogues are oxadiazole, thiadiazole and triazole peptoids. Without limiting to AAT and peptide derivatives of AAT, compounds such as oxadiazole, thiadiazole and triazole peptoids are preferred.
The preferred doses for administration can be anywhere in a range between about 10 ng and about 10 mg per ml of biologic fluid of treated patient. The therapeutically effective amount of AAT peptides or drugs that have similar activities as AAT or peptide drug can be also measured in molar concentrations and may range between about 1 nM and about 1 mM per ml of biologic fluid of treated patient.
It is another object of the present invention to provide pharmaceutical compositions with serine protease inhibiting activity comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one of the compounds of the present invention or a pharmaceutically acceptable salt or prodrug form thereof.
Other viral infections are contemplated to be treated wherein such viral infections are caused/facilitated by a deficiency in AAT levels or by a dysfunction of AAT. Clinical conditions and viral infections resulting from uncontrolled serine protease activity are also within the scope of the present invention and will be treated alike.
A general method of treating a mammal suffering from a pathological condition that is mediated by endogenous serine protease (SP) or SP-like activity is contemplated as well, which comprises administering a therapeutically effective amount of a substance exhibiting mammalian α1-antitrypsin (AAT) or AAT-like activity. This pathological condition can be caused at least in part by a viral infection.
Without limiting to AAT a compound of choice may be one that inhibits proteinase-3, cathepsin G, or elastase.
In accordance with this invention, there is provided a novel class of chemical compounds that are capable of inhibiting and/or blocking the activity of the serine protease(s), which halts the proliferation of a virus including HIV, pharmaceutical compositions containing these compounds, novel intermediates for compounds which inhibit and block the activity of the HIV facilitating serine protease, novel methods for making such compounds, and use of the compounds as inhibitors of the HIV.
It should be apparent that in addition to these preferred embodiments a method is contemplated which consists of treating an individual having a physiological condition caused, in whole or part, by virus shedding. In accordance to this embodiment a method of inhibiting virus release is provided wherein the target of the therapy is a cell and one will contact such cell with an effective amount of a compound having AAT activity.
It is another object of the present invention to provide a novel method for treating HIV infection which comprises administering to a host in need thereof a therapeutically effective combination of (a) one of the compounds of the present invention and (b) one or more compounds selected from the group consisting of HIV reverse transcriptase inhibitors and HIV protease inhibitors. Accordingly reverse transcriptase inhibitor can be selected from a group including nucleoside RT inhibitors: Retrovir (AZT/zidovudine; Glaxo Wellcome); Combivir (Glaxo Wellcome); Epivir (3TC, lamivudine; Glaxo Wellcome); Videx (ddI/didanosine; Bristol-Myers Squibb); Hivid (ddC/zalcitabine; Hoffmann-LaRoche); Zerit (d4T/stavudine; Bristol-Myers Squibb); Ziagen (abacavir, 1592U89; Glaxo Wellcome); Hydrea (Hydroxyurea/HO; nucleoside RT potentiator from Bristol-Myers Squibb) or Non-nucleoside reverse transcriptase inhibitors (NNRTIs): Viramune (nevirapine; Roxane Laboratories); Rescriptor (delavirdine; Pharmacia & Upjohn); Sustiva (efavirenz, DMP-266; DuPont Merck); Preveon (adefovir dipivoxil, bis-POM PMEA; Gilead). Protease inhibitors (PI's) are selected from Fortovase (saquinavir; Hoffmann-La Roche); Norvir (ritonavir; Abbott Laboratories); Crixivan (indinavir; Merck & Company); Viracept (nelfinavir; Agouron Pharmaceuticals); Angenerase (amprenavir/14IW94; GlaxoWellcome), VX-478, KNI-272, CGP-61755, and U-103017.
Also contemplated is a method of preventing acquired or congenital deficiency of functional endogenous AAT levels in a patient susceptible to a viral infection that is mediated by endogenous host serine protease (SP) or SP-like activity by treating the patient with a pharmaceutical composition in a pharmaceutically acceptable carrier comprising an effective amount of a substance exhibiting mammalian α1-antitrypsin (AAT) or AAT-like activity and a thrombolytic agent such as tissue plasminogen activator, urokinase, streptokinase, or combinations or complexes thereof. The pharmaceutical composition may be a peptide or a small molecule, which exhibits AAT or AAT-like activity.
The treatment and prevention of virus induced tumors by administering α1-antitrypsin (AAT) or a compound with AAT-like activity is another object of this invention. Yet another preferred embodiment of this invention is to provide α1-antitrypsin (AAT) or a compound with AAT-like activity for types of cancer that may or may not be virus induced but are capable of metastasizing due to SP activity. Such tumors may comprise fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, rhabdosarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, myeloma, lymphoma, and leukemia.